In developed societies, age-related macular degeneration (AMD) is the most common cause of legal blindness in people over age 65. Emerging evidence demonstrates an important role for retinal pigment epithelium (RPE) mitochondrial dysfunction in dry AMD pathobiology. Mitochondrial dysfunction is characterized by chronic overproduction of superoxide and diminished production of ATP, and it promotes activation of injury response pathways in RPE cells, leading to sub-RPE deposit formation (precursors of lipid-rich drusen) and dysregulation of extracellular matrix turnover, the hallmark features of dry AMD. Thus, mitochondrial-targeting drugs are emerging as an attractive class of potential therapeutics for dry AMD. Technologies to identify the presence of RPE mitochondrial dysfunction in living eyes are limited. Detecting mitochondrial dysfunction in vivo would enable selection of individuals who might benefit from mitochondria-targeting drugs and would accelerate mechanistic studies of mitochondrial dysfunction in dry AMD disease models. The goal of this project is to develop RPE flavoprotein autofluorescence as an imaging biomarker for mitochondrial dysfunction. Mitochondrial dysfunction is accompanied by an increased ratio in the oxidized to reduced forms of flavoproteins (especially FAD+), which are important components of the electron transport chain for ATP production. Increase in oxidized flavoprotein produces a characteristic shift in the autofluorescence emission spectra, which is easily measured in isolated cells or tissues. However, detecting this shift noninvasively in tissues of living animals or humans, especially the eye, is much more challenging. It requires both a specialized device to image the RPE and an analytical approach to detect the specific autofluorescence signature attributable to flavoprotein. In this project, we will design and build prototype multispectral imaging device using a rapidly tunable excitation laser source, an acousto-optical tunable barrier filter, and a confocal scanning laser ophthalmoscope retinal imager, integrated with analytical software based on synchronous fluorescence spectroscopy, to detect and quantify RPE flavoprotein autofluorescence. We will use cell culture and animal model experimental systems to develop and validate this technology, and we will adapt the integrated multispectral imaging device to initiate a pilot clinical study of RPE flavoprotein autofluorescence in human subjects without retinal disease and in subjects with AMD.